Fig. 2

Milder fixation, longer permeabilization, and incubation with antibody gave optimum Bruchpilot staining. Squash preparation of VNC of Drosophila 3rd larval instar. a–c Bruchpilot staining with different combinations of incubation periods for post-fixation and antibody (Ab) incubation with 0.3% PT-X permeabilization for 10 min. d Milder post-fixation for 10 min with 10 min 0.3% PT-X permeabilization. e 20 min 0.3% PT-X permeabilization with milder post-fixation for 10 and 60 min Ab incubation. f 20 min 0.3% PT-X permeabilization and 30 min 0.1% PBT-X blocking with milder post-fixation for 10 and 60 min Ab incubation. Magnification ×40 oil objective, N.A. = 1.3; Scale bars: 25 µm. The experiments were performed using a set of 5–10 isolated VNCs, and the images represent the majority observation amongst each set